SUMMARYWe have examined the
tissue-specific mRNA expression pattern of androgen receptor (AR), both
estrogen receptor (ER)
subtypes ER-alpha and ER-beta and progestin receptor (PR) in 10 bovine
gastrointestinal compartments. Goal of this study was to evaluate
the deviating tissue sensitivities and the influence of the estrogenic
active preparation Ralgro® on the compartment-specific expression
regulation. Ralgro®contains Zeranol which
shows
strong estrogenic and anabolic effects. Eight heifers were treated for
8 weeks with Ralgro® at different dosages (0, 1, 3, and 10 times).
To quantify the very lowabundant steroid eceptormRNAtranscripts
sensitive and reliable real-time (kinetic) reverse transcription
(RT)-PCR quantification methods were validated on the LightCycler.
Expression results indicate the existence of AR and both ER subtypes in
all 10 gastrointestinal
compartments. PR receptor was expressed at very low abundancy.
Gastrointestinal
tissues exhibit a specific ER-alpha and ER-beta expression pattern with
high expression levels for both subtypes in rectum, colon and
ileum.With
increasing Zeranol concentrations a significant down-regulation for
ER-alpha and ER-beta was observed in jejunum (P < 0 .001 and
P<0.05,
respectively). Significant up-regulations under estrogen treatment
could be shown in abomasum for ER-alpha (P < 0.05) and in rectum for
ER-beta (P < 0.001).The authors conclude,
that especially estrogens and the
expression of their corresponding receptor subtypes may play an
important role in the modulation and regulation in gastric as well as
gut functions, cell proliferation and possibly in the pathophysiology
of cell cancer. The different expression patterns of ER-alpha and
ER-beta can be regarded as support of the hypothesis that the subtype
proteins may have different biological functions in the
gastrointestinal tract. AR and PR seem to be not estrogen dependent.

Expression
and localisation of oestrogen and progesterone receptors in thebovine
mammary gland during development, function and involution.

It is now well established that oestrogen and progesterone
are absolutely essential for mammary gland development.
Lactation can be induced in non-pregnant animals by sex steroid hormone
treatment. Most of the genomic actions
of oestrogens are mediated by two oestrogen receptors (ER)-alpha and
ERbeta, and for gestagens in ruminants
by the progesterone receptor (PR). Our aim was the evaluation of mRNA
expression and protein (localisation and
Western blotting) during mammogenesis, lactogenesis, galactopoiesis
(early, middle and late) and involution
(8, 24, 28, 96-108 h and 14-28 days after the end of milking) in
the bovine mammary gland (total no. 53).
During these stages, the mRNA was assessed by means of real-time RT-PCR
(LightCycler). The protein for
ERalpha, ERbeta and PR was localised by immunohistochemistry and
Western blotting. The mRNA expression
results indicated the existence of ERalpha, ERbeta and PR in bovine
mammary gland. Both ERalpha and
PR are expressed in fg/ micro g total RNA range. The highest mRNA
expression was found for ERalpha and
PR in the tIssue of non-pregnant heifers, followed by a significant
decrease to a lower level at the time
of lactogenesis with low concentrations remaining during lactation and
the first 4 weeks of involution.
In contrast, the expression of ERbeta was about 1000-fold lower (ag/
micro g total RNA) and showed no
clear difference during the stages examined, with a significant
increase only 2-4 weeks after the end of milking.
Immunolocalisation for ERalpha revealed a strong positive staining in
nuclei of lactocytes in non-pregnant
heifers, became undetectable during pregnancy, lactogenesis and
lactation, and was again detectable 14-28
days after the end of milking. In contrast, PR was localised in the
nuclei of epithelial cells in the mammary tIssue
of non-pregnant heifers, in primigravid animals, and during late
lactation and involution. During lactogenesis,
peak and mid lactation, fewer nuclei of epithelial cells were positive,
but increased staining of the cytoplasm of
epithelial cells was obvious. ERalpha and ERbeta protein was found in
all mammary gland stages
examined by Western blotting. In contrast to mRNA expression, the
protein signal for ERalpha was
weaker in the tIssue of non-pregnant heifers and during involution (4
weeks). ERbeta protein showed a stronger signal
(two isoform bands) in non-pregnant heifers and 4 weeks after the end
of milking. This correlated with the mRNA expression
data. Three isoforms of PR (A, B and C) were found by Western blotting
in the tIssue of non-pregnant heifers, but only
isoform B remained during the following stages (lactogenesis,
galactopoiesis and involution). In conclusion, the mRNA
expression and protein data for ER and PR showed clear regulatory
changes, suggesting involvement
of these receptors in bovine mammary gland development and involution.

Expression
of estrogen and progesterone receptors in the bovine
ovary during estrous cycle and pregnancy.

Berisha B, Pfaffl
MW, Schams D.Endocrine
2002 Apr;17(3):207-214

The
objective of the study was to demonstrate the mRNA expression of
estrogenreceptor
a (ERa), ERbeta, and
progesterone receptor (PR) by block reversetranscription-polymerase
chain reaction(RT-PCR) and real-time RT-PCR(LightCycler)
in bovine ovarian follicles and in corpus luteum during theestrous
cycle and pregnancy. The mRNA expression of ERalpha and ERbeta mRNA
intheca interna
tissue (TI) (lower
pg/microg RNA) increased continuously andsignificantly
during final growth of follicles, with much higher levels forERalpha.
The mRNA expression of ERalpha and ERbeta in granulosa cells (GC)(fg/microg
RNA) increased continuously during follicle growth but without anysignificant
change. The expression of mRNA for PR in follicles (lower fg/microgRNA)
increased continuously to maximum level in preovulatory follicles with asignificant
change only in TI. The highest mRNA expression for ERalpha(fg/microg
RNA) was detected in corpus luteum (CL) during the early lutealphase,
following by a significant decrease of expression during the mid, late,and
regression phases. In contrast, ERbeta mRNA expression is relatively
highduring the
early stage, decreased
during the late
early and mid luteal phase,and
increased significantly again during the late luteal phase and after CLregression.
During pregnancy (>3 mo), low levels of ERalpha and ERbeta mRNAexpression
(<25 fg/microg RNA) with no significant changes were measured.
Nosignificant
change in PR mRNA
expression
(levels <13 fg/microg RNA) during theestrous
cycle and pregnancy in bovine CL were found. The results suggest
anautocrine/paracrine
role of steroid
receptors in the regulation of finalfollicle
growth and corpus luteum formation and function.

We have examined the
tissue specific mRNA expression of ERa and ERb in various bovine tissues using real-time
RT-PCR. Goal of this study was to evaluate the deviating
tissue sensitivities
and the influence of the estrogenic active preparation RALGRO on the tissue
specific expression and regulation of both ER subtypes. RALGRO contains Zeranol
(a-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong
estrogenic and anabolic effects, and exhibits all symptoms of hyper-estrogenism in particular
reproductive and developmental disorders. Eight heifers were treated over 8 weeks with
multiple dose implantations (0x, 1x, 3x, 10x) of Zeranol.Plasma Zeranol
concentration, measured by enzyme-immuno-assay, of multiple treated heifers Zeranol were
elevated. To quantify ERa and ERb transcripts also in low abundant tissues, sensitive
and reliable real-time RT-PCR quantification methods were developed and validated on
the LightCycler. Expression results indicate the existence
of both ER subtypes
in all 15 investigated tissues. All tissue exhibit a specific ERalpha and ERbeta expression
pattern and regulation. With increasing Zeranol concentrations
a significant
down-regulation of ERa mRNA expression could be observed
in jejunum
(p<0.001)
and kidney medulla (p<0.05). These data support the hypothesis, that the
ERb may have different biological functions than ERa, especially in kidney and the jejunum.

Synthetic
progestagen like melengestrol acetate (MGA) are widely used for estrus
synchronisation and for growth promotion in
cattle production. The metabolic
effects exceed its primary potency as a
progestagen. It is
speculated that MGA stimulates follicle development and
thereby endogenous estrogen production,
but inhibits ovulation. To
investigate the dose dependent effects on the
mRNA expression
levels, six heifers were fed during 8 weeks with different levels
of MGA
(0.5 mg, 1.5 mg, 5 mg) daily and two heifers served as control. The
expression of steroid receptor mRNA
[androgen receptor (AR), progesterone receptor
(PR), estrogen receptor (ER) ERa and ERb],
insulin-like growth factor-1 (IGF-1) and its
receptor were quantified in liver, neck
(m. splenius) and shoulder
muscularity (m. deltoideus). Plasma concentrations of IGF-1 were quantified by
radioimmunoassay. In treated animals the MGA plasma levels were elevated over the complete
treatment period, corresponding to the MGA treatment concentrations. IGF-1 concentrations of control
animals were at constant levels. Plasma levels for estradiol (E2) and IGF-1 were increased in low
MGA treatment group. Overdosed MGA decreased progesterone (P4) and E2 levels. To quantify
the IGF-1 and all receptor mRNA transcripts, sensitive and reliable real-time RT-PCR quantification
methods were developed and validated in
the LightCycler. A dose dependent
relationship between
increasing MGA concentrations and mRNA expression were observed in liver for AR and IGF-1
receptor, and in neck muscularity for IGF-1. ERa in liver and neck
muscle showed
a trend of increasing expression.

The effect of
testosterone on sexual dimorphism is evident by differentialgrowth of forelimb
and neck muscles in bulls and steers. Divergent hormonesensitivites may account for
the differential growth rates of individualmuscles.
Therefore, the
objective of this study was to compare
androgen receptor(AR) expression in three
different muscles of bulls and
steers
at various agesand growth rates. Thirty
Montbeliard bulls and 30 steers
were assigned to fourslaughter age groups.
Four or five animals of each sex were
slaughtered at 4 and8 mo of age. Animals in
the remaining two slaughter groups
(12 and 16 mo) weredivided into groups of
either restricted (R) or ad libitum
(AL) access to feed.Five animals of each sex
and diet were slaughtered
at the end of the restrictedintake period at
12 mo of age. To simulate compensatory
growth, the remaininganimals (R and AL) were
allowed ad libitum access
to feed until slaughter at 16mo of age. Total RNA
was extracted from samples of
semitendinosus (ST), tricepsbrachii (TB), and
splenius (SP) muscles. Androgen receptor
mRNA was quantifiedin 200-ng total RNA
preparations using an internally
standardized reversetranscription (RT) PCR
assay. Data were analyzed using 18S
ribosomal RNAconcentrations as a covariable.
Steers had higher AR mRNA
levels per RNA unitthan bulls (P <
.01). Androgen receptor mRNA levels differed between muscles (P< 0.05), with
lowest expression in the SP. The pattern of AR expression differed(P < 0.05) for
each muscle with increasing age. Between 4 and 12 mo of age, ARmRNA levels
increased (P < 0.05) in SP but remained unchanged in the ST
and TB.Feeding
regimen had no effect on muscle AR expression, but steers exhibitingcompensatory growth
had higher AR mRNA levels than AL steers (P < 0 .01) or bulls(P < 0 .01).
Our results show that AR expression is muscle-specific and may bemodulated by
circulating testicular hormones. These data suggest that theregulation of AR
expression may be linked to allometric muscle growth patternsin cattle and
compensatory gain in steers.

Quantification
of androgen receptor mRNA in tissues bycompetitive
co-amplification of a template in RT-PCR

We describe a
polymerase chain reaction (PCR)-based method for thequantification of androgen
receptor (AR) mRNA in tissues. The amount of PCRproducts
depends on the
exponential amplification of the initial cDNA copynumber; therefore minor
differences in the efficiency of amplification maydramatically influence the
final
product yield. To overcome these tube-to-tubedifferences
in reaction
efficiency, an internal control AR cRNA was reversetranscribed along with the
target mRNA using the same primers. This standard wasobtained by deleting
a 38 bp fragment from an amplified bovine AR sequence,which was then subcloned and
transcribed into cRNA. Known dilutions of thecompetitor
cRNA were spiked
into a series of RT-PCR reaction tubes containingequal amounts of the target
mRNA. Following RT-PCR, the co-amplified specimensobtained were separated by gel
electrophoresis and quantified by densitometricanalysis of ethidium
bromide stain. We applied this method to quantify theAR-mRNA in skeletal muscle of
castrated as well as from intact male cattle. Theapplicability of the
quantification system for AR-mRNA described herein wasdemonstrated for other species,
e.g. man.

Expression
of estrogen and androgen receptor in the bovine
gastrointestinal tract

Reproductive and
maturational nutritive needs are
examples for situations inwhich alterations in
circulating concentrations of sex
steroids are associatedwith changes in
gastrointestinal function. In order to
investigate whether thereis a causal
relationship between sex
steroids and gastrointestinal function, weaimed
to investigate the
responsiveness for androgens and for estrogens of thebovine gastrointestinal tract.
Using Northern blot analysis, estrogen receptor(ER)
mRNA was detected in rumen
tissue. Comparing the ER expression in rumenfrom
females of different
reproductive stages, we found that no differencesrelated to cycle stage,
pregnancy or parturition could be detected. In contrast,the ER expression rates in the
uterus of the respective animals showed the samedependency
of reproductive
stage as demonstrated earlier for the ER protein,indicating that there might be
a tissue
specific regulation of ER. By in-situhybridization
of rumen tissue
sections the expression of ER was localized in theepithelium of the papillae. In
the muscular layer no positive signals for ERmRNA
were observed. Above
rumen, the
presence of ER and androgen receptor (AR)mRNA was determined
in various intestinal tissues using reverse transcription(RT) and polymerase
chain reaction (PCR). Primers were selected from the bovineandrogen and
estrogen receptor sequence to amplify parts of the sequence codingfor the hormone
binding part of the respective receptor. The PCR amplifies weresubsequently
electrophoresed on 1% agarose gels and visualized by ethidiumbromide staining. ER
mRNA expression was demonstrated in reticulum, omasum,abomasum, duodenum, jejunum,
ileum, caecum and colon. AR mRNA expression was notdetermined in the
forestomaches, but was present in all intestinal segments investigated.

Uterine
Androgen Receptor mRNA Expression in Metestrous and Anestrous
Bitches being healthy or suffering from pyometra

SummaryThe importance of
androgens for the female reproductive system has beeninvestigated for decades and a
number of androgen sensitive processes has nowbeen
identified in female
reproductive organs. For carnivore species no datawere available so far about
uterine androgen sensitivity and its regulation. Thepresent study therefore aimed
to investigate whether androgen receptors (AR) arepresent in the dog uterus,
whether they are regulated throughout the ovariancycle and whether pyometra
affects their expression rate. Uterine tissue sampleswere collected from 28 bitches
of different ages and various breeds. The sampleswere grouped according to the
stage of estrous cycle (metestrus ME or anestrusAE)
and the pathological status
of the uterus (i.e. suffering from pyometra ornot).
Androgen receptor mRNA
(AR mRNA) was quantified from 500 ng of total RNAisolated from the tissue
samples using
an internally standardized reversetranscription
polymerase chain
reaction (RT-PCR) described previously. Theamount
of total RNA extractable
per g tissue was elevated during pyometra. Thesuccessful
amplification of the
expected 172 bp fragment from canine uterine RNAtogether with the
confirmation of the identity of this fragment by sequenceanalysis,
demonstrates that AR is expressed in this particular tissue. Comparingthe expression rates
in uteri from bitches during ME or AE being healthy (H) orsuffering from
pyometra (P), the only significant (p < 0.01) difference wasfound between H and
P uteri during ME with 3.5-fold lower expression rates in P.Although the same
seems true for AE bitches, a significant difference could notbe demonstrated due
to the low number (n = 2) of diseased animals in the AEgroup. There was no evident
effect of the stage of ovarian cycle on uterine AR mRNA levels.

SUMMARYAlteration of
androgen receptor function due to hormonally active compounds in the
environment, may be responsible for impaired reproductive function in
aquatic wildlife. Based on human prostate carcinoma 22RV1 cells, a cell
culture expression system was established to test effects of putative
androgenic/antiandrogenic compounds on endogenous gene expression.
22RV1 cells were shown to express human androgen receptor, but not
human progestin (hPR) or human oestrogen receptor (hER) alpha and beta.
Six androgen-regulated genes (ARGs) were chosen to determine
androgenic/antiandrogenic action using highly sensitive real-time
RT-PCR. Results showed that gene expression is altered in a
time-dependent manner. After stimulation of cells by DHT (10 nM),
synthetic androgen R1881 (1 nM), or organic pesticides (difenoconazole,
fentinacetate, etramethrin) TMPRSS2 mRNA expressionwas down-regulated
by the factor 0.6 after 24 h ofDHTtreatment.Similar
results were obtained
when cells were assayed for mRNA expression of PSA after fentinacetate
and R1881 stimulation. In contrast, TMPRSS2 expression was up-regulated
by the factor 0.9 when cells were stimulated by tetramethrin. Final
goal of the work is a sensitive determination of differential gene
expression by different compounds under study, achievement of
substance-specific expression patterns and function related analysis of
potential androgens/antiandrogens.

Detection
and quantification of mRNA-expressionof alpha- and beta-adrenergic
receptor subtypesin the
mammary gland of dairy cows

SummaryAdrenergic receptors
are pharmacologically classified into the receptor types alpha-1,
alpha-2, beta-1, beta-2, and
beta-3. Structural differences and varying affinities in radioligand
binding studies lead to a further classification of alpha-1- and
alpha-2-receptors into subtypes which are termed alpha-1A (formerly C),
alpha-1B, and alpha-1D-(formerly AD), and alpha-2AD, alpha-2B,
and alpha-2C, respectively. mRNA-expression of all but one
alpha-adrenergic receptor subtypes and of all beta-adrenergic receptor
types was measured quantitatively in total RNA extracted from mammary
tissue of ten lactating dairy cows by real-time reverse transcriptase
(RT) polymerase chain
reaction (PCR). mRNA expression of alpha1-adrenergic receptors was
highest for the alpha-1A-subtype followed by alpha-1B, whereas the
alpha-1D-subtype could not be detected. The highest mRNA expression of
alpha-2-adrenergic receptors was found for the alpha-2AD-subtype,
followed by alpha-2B and alpha-2C. Within the beta-adrenergic
receptors, the beta-2-receptor type was most highly expressed, followed
by beta-1 and beta-3. In conclusion, eight of nine adrenergic receptors
classified to date were detected and relatively quantified in the
mammary gland of dairy cows.

New
sequences of alpha-adrenergic receptor subtype mRNA at
GENBANK and EMBL

Milking
characteristics and
their relation to adrenergic receptor mRNA expression
and ligand binding in the mammary gland of dairy cows

Stimulation of - and -adrenergic receptors in the
bovine mammary gland affects milking characteristics such as milk yield
and peak flow rate. The aim of this study was to detect possible
correlations between milkability, adrenergic receptor binding capacity
and receptor expression at the mRNA level. In addition, dose–response
relationships of - and -adrenergic receptor stimulation
were evaluated after application of an - and -adrenergic receptor agonist,
respectively in different
dosages. Density and distribution of adrenergic receptor binding
sites in the region around the large mammary ducts were investigated
as well as adrenergic receptor mRNA expression. Milk flow of
one-quarter
was recorded in 10 cows without or with additional - and -adrenergic receptor stimulation
in three dosages each.
After slaughter, mammary tissue was taken from the region around
the large mammary ducts in the previously investigated quarters.
Protein
and RNA were extracted for measuring 1-, 2-, and 2-adrenergic receptor
binding sites and mRNA expression levels by real-time polymerase chain
reaction (RT-PCR). Peak flow rate without additional adrenergic
receptor stimulation was negatively correlated with 2-adrenergic receptor
binding (maximal
binding capacity, Bmax) and positively correlated
with 2-adrenergic receptor
expression at the mRNA level (crossing point (CP) of the real-time
PCR). During -adrenergic receptor stimulation,
there was a positive correlation between milkability and 2-adrenergic receptor
mRNA expression,
whereas during -adrenergic receptor stimulation
no correlations were detected. Dose–response relationships were
existing during -adrenergic receptor
stimulations, but not during -adrenergic receptor stimulations
at four dosages each including control milking. Significant changes in
milk yield and peak flow rate mainly occurred after application of an -adrenergic receptor agonist. In
conclusion, high mRNA expression levels or binding capacities of
adrenergic receptors do not necessarily lead to according reactions in
vivo, concerning milk yield and peak flow rate. To influence milking
characteristics, individual reactions of the cow on adrenergic
stimulation have to be considered.

The
mRNA expression of the
members of the IGF-system
in bovine corpus luteum during induced luteolysis

The components
of the IGF-system were shown to be differentially regulated in bovine
antral
follicles and corpora lutea (CL) during different stages of the estrous
cycle, and to have impor-
tant functions for specific stages. The aim of this study was to
investigate the detailed pattern
of mRNA expression of most constituents of the IGF-system and their
possible involvement in
prostaglandin (PG)F2-alpha-induced luteolysis in the bovine CL.
Therefore, cows in the mid-luteal
phase (days 8–12) were injected with the PGF2-analogue Cloprostenol,
and CL were collected by
transvaginal ovariectomy at 2, 4, 12, 48 and 64 h after
PGF2-injection. Real-time RT-PCR using
SYBR Green I detection was employed to determine mRNA expressions of
the following factors:
ubiquitin (UBQ), insulin-like growth factor I (IGF I), IGF II,
IGF-receptor type 1 (IGFR-1), growth
hormone receptor (GH-R) and IGF-binding proteins-1–6 (IGFBP-1–6). Total
extractable RNA de-
creased with ongoing luteolysis. IGFBP-1 mRNA was significantly
up-regulated at 2 h after PGF2-alpha
and maximal at 4 h with a 34-fold increase. IGFBP-5 mRNA was
significantly up-regulated after
12 h with a maximum of an 11-fold increase at 64 h. For GH-R, IGFR-1,
IGF II, IGFBP-3 and -4
mRNA expression, we found a significant down-regulation in certain
stages. There was a significant
up-regulation for IGFBP-2 and -6 mRNA at 64 h after induced luteolysis.
There were no significant
changes in IGF I mRNA expression. In conclusion, the IGF-system with
all its components seems
to play an important role in the very complex process of
PGF2-alpha-induced luteolysis in bovine CL.

SummaryReverse
transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for
analyzing mRNA in extremely low abundance. Real-timeRT-PCR using SYBR Green I
detection combines the ease and necessary exactness to be able to produce
reliable as well as rapid results. To obtain highly accurate and
reliable results in a real-time RT-PCR a
highly defined calibration
curve is needed. We designed and developed
nine different
calibration curves, based on recombinant DNA plasmid standards and established them on a
constant real-time PCR platform for the following factors: growth hormone receptor
(GHR), insulin-like growth factor (IGF)-1, IGF-1 receptor (IGF-1R),
IGF-2, IGF-2
receptor (IGF-2R), insulin receptor (INSR), and IGF-binding proteins
(IGF-BP) 1, 2 and 3. Developed assays were
applied
in the LightCycler system on bovine ileum and liver total RNA and showed high specifity
and sensitivity of quantification. All assays had a detection limit of under 35 recombinant
DNA molecules present in the capillary. The SYBR Green I determination resulted in a
reliable and accurate quantification with high test linearity (Pearson correlation
coefficient r > 0.99) over seven orders of magnitude from <102 to
>108 recombinant
DNA start molecules and an assay variation of maximal 5.3%.
Applicability of the method was shown by
analyzing mRNA levels in newborn calves:
mRNA concentrations per gram tissue of
mRNAs of IGF-1, IGF-1R, IGF-2, IGF-2R, GHR, INSR, and IGFBP-1, -2 and -3 were all
different between in liver and ileum and the traits all exhibited
individual differences.

Abundance of
message for
insulin-like growth factors-I and -II and for receptors for growth
hormone, insulin-like growth factors-I and -II, and insulin in the
intestine and liver of pre- and full-term calves

The
somatotropic axis and insulin are involved in pre- and postnataldevelopment.
In pre- and full-term calves (GrPo and GrNo; bornafter 277 and 290 d of pregnancy, respectively) and in
pretermcalves on d 8 of life after being fed for 7 d (GrP8),
we studiedwhether there are differences
in the abundance of messengerRNA (mRNA) of IGF-I and
IGF-II and of receptors for GH, IGF-I,IGF-II, and
insulin among different intestinal sites (duodenum,jejunum,
ileum, and colon) and whether there are ontogeneticdifferences
during the perinatal period in intestine and liver.Intestinal
site differences (P < 0.05) existed in mRNA levelsof
IGF-I and IGF-II and receptors for GH, IGF-I, IGF-II, andinsulin.
Abundance of mRNA of IGF-I and -II and of receptorsfor
IGF-I and GH was highest (P < 0.05) in the colon, abundanceof the receptor for IGF-II was comparably high in the colonand ileum, and that of the receptor for insulin was
similarlyhigh in colon, ileum, and jejunum. Among GrPo,
GrNo, and GrP8groups, there were
differences (P < 0.05) in mRNA levelsof IGF-I
and IGF-II and of receptors for GH, IGF-I, IGF-II andinsulin.
Abundance of mRNA
of IGF-I and IGF-II and of receptorsfor GH, IGF-I, IGF-II
and
insulin was highest (P < 0.05)in GrPo
calves
immediately after birth and was primarily seenin the
ileum.
In liver, the mRNA levels differed (P < 0.05)among
groups for IGF-II and receptors for IGF-I, IGF-II, andinsulin,
and were highest (P < 0.05) for IGF-II in GrPo,for receptors of IGF-I in GrNo, and were higher (P
< 0.05)in GrPo than GrP8 for
receptors
of IGF-II. In conclusion, mRNAlevels of IGF-I and IGF-II
and of receptors for GH, IGF-I, IGF-II,and insulin were
different
at different intestinal sites andin intestine and liver
and
changed during the perinatal period.

SUMMARYIntestinal
development is modified by age and nutrition, mediated in part by
insulin-like growth factors (IGF-I,IGF-II) and
insulin.
We have investigated whether expression of IGF-I, IGF-II and
insulin receptors (IGFIR,IGF-IIR and IR;
measured by real-time RT-PCR) and binding
capacity (Bmax) of IGF-IR, IGF-IIR and IRin
the mucosa
of the small and large intestine of neonatal calves are modified
by age and di?erent feeding regimes. In experiment 1, pre-term (GrP)
and full-term (GrN) calves (after 277 and 290 days of pregnancy
respectively) were killed immediately after birth before being fed;
a further group of full-term calves were fed for 7 days and killed
on day 8 of life (GrC1–3). In experiment 2, full-term calves were
killed on day 8 after being fed first-colostrum for 7 days (GrCmax),
colostrum of the first six milkings for 3 days (GrC1–3) or
milk-based formula for 3 days (GrF1–3). Intestinal sites di?ered with
respect to expression levels of IGF-IR (duodenum>jejunum in GrC1–3;
ileum>colon, duodenumjejunum in GrF1–3), IGF-IIR (colon>duodenum
and ileum in GrN), and IR (lowest in ileum in GrP and CrN; highest in
colon in GrC1–3 and GrCmax). They also differed with respect to
Bmax of IGF-IR (ileum and colon>duodenum and jejunum in GrP; ileum
and colon>jejunum in GrN; colon>jejunum in GrC1–3; lowest in
jejunum in GrF1–3), IGF-IIR (duodenum and colon>jejunum and ileum
in GrP; duodenum>ilem and colon>jejunum in GrN; duodenum, jejunum
and colon>ileum in GrCmax, GrC1–3, and GrF1–3) and IR
(ileum>duodenum, jejunum and colon in GrCmax, GrC1–3, and GrF1–3).
There were significant differences between groups in the expression of
IGF-IR (GrF1–3> GrCmax and GrC1–3 in ileum), IGF-IIR (GrN>GrP and
GrC1–3 in colon; GrN>GrC1–3 in jejunum and total intestine), and IR
(GrCmax>GrF1–3 in colon) and in the Bmax of IGF-IR (GrP>GrN in
colon; GrCmax>GrF1–3 in jejunum), IGF-IIR (GrN>GrP in duodenum,
ileum and total intestine; GrN>GrC1–3 in duodenum, ileum, colon and
total intestine) and IR (GrN>GrP in total intestine;GrC1–3>GrN in ileum and
total intestine). In addition, Bmax values of IGF-IR, IGF-IIR and IR
were correlatedwith villus circumference,
villus height/crypt depth and
proliferation rate of crypt cells at
various intestinal sites.There were marked
differences in Bmax of IGF-IR, IGF-IIR and
IR dependent on mRNA levels, indicatingthat differences in
Bmax were the consequence of differences in posttranslational control
and of receptor turnoverrates. In conclusion
IGF-IR, IGF-IIR and IR expressions and Bmax in intestinal mucosa
were different at differentintestinal sites
and were variably affected by age, but not
significantly affected by differences
in nutrition.Receptor densities were
selectively associated with
intestinal mucosa growth.

Quantification
of insulin-like growth factor-1 (IGF-1) mRNA: development
and validation of an internally standardised competitive RT-PCR

SummaryTo investigate the
role of local IGF-1 mRNA expression in various tissues, wedeveloped and
validated a method which allows for a specific, sensitive andreliable
quantification of IGF-1 mRNA: an internally standardised ReverseTranscription-Polymerase
Chain
Reaction (RT-PCR). A synthetic competitivetemplate
IGF-I standard cRNA
(IGF-1 cRNA) was designed, which contains the sameflanking primer sequences used
to amplify the wild type IGF-1 mRNA, but differsby
56 bp in length.
To obtain the IGF-1 mRNA concentration present in tissue RNAsamples, series of
250 ng total-RNA were spiked with three known quantities ofthe standard IGF-1
cRNA, incubated for competitive RT-PCR reactions and the twoamplificates
obtained (184 bp from IGF-1 cRNA and 240 bp from the wild typeIGF-I mRNA) were
subsequently separated and quantified by HPLC-UV. For everyindividual tissue
RNA
sample, the ratio R (R = competitor PCR product / wildtype PCR product) was
plotted against the number of starting molecules of thecompetitor IGF-1 cRNA.
The initial amount of IGF-1 mRNA present in the samplecan then be read off where
R = 1. The validated assay had a detection limit of1600 IGF-1 cRNA
molecules/reaction, the intra-assay variation was 7.4% (n = 5)and linearity (r =
0.997) was given between 140 ng to 840 ng total-RNA input.The present method
was first applied to study the effect of long term castrationon the IGF-1
expression rates in bovine
tissues. The hepatic IGF-1 mRNAconcentrations
were well
correlated (r = 0.81) with the plasma concentrations as quantified by RIA
and were higher in intact than in castrated animals. In twoskeletal muscles (m.
splenius and m. gastrocnemius) IGF-1 mRNA concentrationswere 20- and 35- times lower
than in liver, respectively, without anydifferences
between
steers and bulls. In bulls, the IGF-1 mRNA expression washigher in m.
splenius (p < 0.01) than m. gastrocnemius, indicating that locallyproduced IGF-1 might
be important for sexually dimorphic muscle growth patterns.

Quantification
of the insulin like growth factor-1 (IGF-1) mRNA:Modulation
of growth intensity by feeding results in inter- and intra-tissue
specific differences of IGF-1 mRNA expression in steer

SummaryThe effect of
constant and compensating body growth
velocities on IGF-1 mRNAexpression was studied
in various tissues of growing steers.
Twenty-six steerswere allocated to three
groups in which the average daily
gains were kept eitherconstantly high on
intensive feeding, low on pasture feeding
or were acceleratedto compensatory growth
after feed restriction. All animals
were slaughtered at570+/-2.6 kg and samples
were collected from liver, heart,
kidney and from 4different muscles (m.
splenius, m. soleus, m. cutaneus
truncii and m.semispinalis capitis), which
were selected in order to include maximaldifferences
in fibre
composition as well as in growth impetus. IGF-1 mRNA wasquantified by a validated
internally standardised RT-PCR method. The amount ofRNA extracted from the various
tissues investigated was constant within eachtype
of tissue and showed no
differences between treatment groups. As indicatedby a constant ratio between the
amount of RNA extracted and the DNAconcentrations,
there was no
effect of the feeding on total transcriptionalactivity.
The order of IGF-1
mRNA abundance per g tissue was liver > > kidney >heart > skeletal
muscle. The different feeding regimen resulted in significantdifferences of IGF-1
mRNA expression rates in all organs showing differentpatterns between organs. IGF-1
mRNA concentrations showed muscle specificdifferences
and also divergent
reactions in response to the differing growthrates.
These results support
that the liver is the main IGF-1 producing tissue;above that they indicate that
skeletal muscle, in particular when taking itsabsolute
mass into account,
might considerably contribute to the IGF-1 levels inblood. Our findings demonstrate
that IGF-1 mRNA expression is regulated tissuespecifically
not only between
different organs but also within musculature.

SUMMARYSynthetic
progestagen like melengestrol acetate (MGA) are widely used for estrus
synchronisation and for growth promotion in
cattle production. The metabolic
effects exceed its primary potencyas a
progestagen. It is
speculated that MGA stimulates follicle development and thereby endogenous estrogen
production, but inhibits ovulation. To investigate the dose dependent effects on the mRNA expression
levels, six heifers were fed during 8 weeks with different levels
of MGA
(0.5 mg, 1.5 mg, 5 mg) daily and two heifers served as control. The
expression of steroid receptor mRNA [androgen
receptor (AR), progesterone receptor
(PR), estrogen receptor (ER) ERa and ERb],
insulin-like growth factor-1 (IGF-1) and its
receptor were quantified in liver, neck
(m. splenius) and shoulder
muscularity (m. deltoideus). Plasma concentrations of IGF-1 were quantified by
radioimmunoassay. In treated animals the MGA plasma levels were elevated over the complete
treatment period, corresponding to the MGA treatment concentrations. IGF-1 concentrations of control
animals were at constant levels. Plasma levels for estradiol (E2) and IGF-1 were increased in low
MGA treatment group. Overdosed MGA decreased progesterone (P4) and E2 levels. To quantify
the IGF-1 and all receptor mRNA transcripts, sensitive and reliable real-time RT-PCR quantification
methods were developed and validated in the LightCycler. A dose dependent relationship between
increasing MGA concentrations and mRNA expression were observed in liver for AR and IGF-1
receptor, and in neck muscularity for IGF-1. ERa in liver and neck
muscle showed
a trend of increasing expression.

Journal of
Huazhong University of Science and Technology 22(1): 69-72.

SummaryPrimary open-angle
glaucoma (POAG) is a leading cause of blindness, which involves optic
neuropathy accompanied by characteristic visual field defects and
is often associated with elevated intraocular pressure due to
disturbance of aqueous humor outflow through the trabecular meshwork
(TM) (1). The pathophysiology of the TM in POAG has been characterized
by an increase in extracellular matrix
components and a decrease in the number of TM cells (2). Polypeptide
growth factors are critical modulators that control normal cell
functions such as proliferation, motility, differentioation,
phagocytosis and
extracellular matrix synthesis and degradation. Studies of secretion
of growth factors which function on TM cells, especially on
proliferation
and extracellular matrix metabolism, are critical to our understanding
of POAG and the development of new antiglaucoma therapy (3). The objective of this study was
to use RT-PCR and immunohistochemistry to determine whether IGF-I is
expressed by TM cells.

Methods.
The reverse transcriptase-polymerase chain
reaction (RT-PCR) was used for detection of IGF-1 mRNA. To detect the
protein on the cells an IGF-1-specific immunohistochemical stain was
used on trabecular meshwork cells.

Results.
A single 240 bp RT-PCR product was obtained, the RT-PCR product was
verified by sequencing and the derived sequence was homologous to the
known bovine sequence. IGF-1 immunostaining was positive in the
cytoplasm of trabecular meshwork cells.

Conclusions. We
conclude that trabecular meshwork cells produce
IGF-1 mRNA and contribute to the presence of IGF-1 protein in the
trabecular meshwork microenvironment as well as aqueous humor.
Trabecular meshwork cells were affected by IGF-1 not only through
paracrine but also through autocrine action. Whether regulations in
IGF-1 production may contribute to the pathoge-nesis of primary
open-angle glaucoma and the possibility of promoting the autocrine
action of IGF-1 by trabecular meshwork cells to treat the disease is
worth further investigation.

Differences
in the somatotropic axis, in blood cortisol, insulin and
thyroid hormone concentrations between two pig
genotypes
with markedly divergent growth rates and the
effects of growth hormone treatment.

SummaryThe intention of the
current study was to gain more insight into the endocrine and molecular
controlmechanisms
of growth in the pig. For this purpose various growth related
parameters were
determinedin
4-month-old barrows of two extreme pig genotyes, the
small, obese Göttingen Miniature (GM) and thelarge and lean German Landrace
(DL). Mean growth hormone (GH) concentration, GH pulse frequencyand GH pulse
amplitude did not differ between breeds. Likewise, plasma IGF-1,
thyroxine,tri-iodothyronine
(T3) concentrations were similar in both breeds. However the plasma GH
response(maximum
level and area under curve) to a single i.v. injection of GHRH in DL
was higher than in GM(P < 0.05).
Furthermore, basal plasma insulin and in
particular plasma cortisol concentrations
werehigher
in GM compared with DL pigs (P < 0.05 and P
<0.01 respectively). Analysis of cortisol during 4-hfrequent blood sampling
indicated higher cortisol amplitudes in GM compared with DL (P
< 0.01).Specific bGH-binding to
hepatic membrane preparations
was not different between breeds and IGF-1 mRNA
concentrations determined
by reverse transcription-polymerase chain reaction in liver, m.semimenbranosus
and m. longissimus dorsi were similar in both breeds. I.m. treatment
with recombinantporcine somatotropin
(rpST; 70 mg/ kg live weight)
over an 8-day period in contemporary barrowsincreased
without any breed
difference, plasma IGF-1, T3 and insulin concentrations and hepaticspecific
bGH-binding, but did not affect thyroxine or cortisol concentrations in
plasma. IGF-1 geneexpression was also elevated
in liver and muscle tissues in
rpST-treated animals without obvious breedeffects.
The observations
underline the complexity of the hormonal and molecular control of
growth andsupport
the notion that differences in growth potential are the consequence of
differences at variouslevels of the
somatotropic axis and apparently relate to
differences in other control systems of energymetabolism
such as the
pituitary adrenal axis or the endocrine pancreas as well.

Isoform A of the human insulin receptor and the IGFreceptor type 2
(IGFR-2) are
both receptors for insulin- like growth factor II (IGF II), which plays
a major
role in luteal development and function in bovine species. The
objective of this
study was to determine if both insulin receptor isoforms and IGFR-2 were
expressed in the bovine corpus luteum (CL) and if they were regulated
during the
estrous cycle, pregnancy, and induced luteolysis. CL were collected at
the
slaughterhouse. For induced luteolysis, CL were obtained by transvaginal
ovariectomy at 2, 4, 12, 48, and 64 h after prostaglandin (PG)
F2alpha-injection. Real-time RT-PCR was applied to investigate mRNA
expression.
Two alternatively spliced transcripts encoding the insulin receptor were
detected in bovine CL. These two isoforms corresponded to the known
isoforms A
(IR-A) and B (IR-B) in humans. IR-A mRNA predominated in bovine CL and
was
significantly down-regulated on d 5-7. IR-B mRNA was significantly
up-regulated
in the late luteal stage and during early pregnancy. IR-A showed a
significant
down-regulation at 48 h after PGF2. IGFR-2 mRNA was significantly
up-regulated
in mid and late luteal stages (d 8-18). It is proposed that the
differential
mRNA expression of IR-A and IGFR-2, both binding IGF II, may play a
role in the
development and function of the bovine corpus luteum.New
sequences of Insulin receptor subtype mRNA at
GENBANK and EMBL

Serotonin
(5-Hydroxytryptamine; 5-HT) is involved in a wide
range of physiological functions and pathological states in humans.
There exists evidence that serotonergic pathways are also involved in
gastrointestinal (GI) motility disorders in ruminants such as displaced
abomasum or cecal dilatation/dislocation. This study aimed to develop
and validate real-time PCR assays for quantitative mRNA analysis of
5-HT receptor subtypes in bovine tissues. Because the bovine
5-HT receptor nucleotide sequences were completely unknown before,
multiple
species (human, mouse, and rat) comparisons of nucleotide sequences
were
done and primers used for bovine cDNA amplification were derived from
human or mouse sequences in highly homologous regions. LightCycler
real-time PCR assays for the following bovine 5-HT receptor subtypes
were developed and validated: 5-HT1a, 5-HT1b, 5-HT1d, 5-HT1e, 5-HT1f,
5-HT2a, 5-HT2b,
5-HT2c, and 5-HT4. Intra- and inter-assay CVs for the 9 established
subtypes ranged from 0.49 to 2.54%. As a first physiological
application,
mRNA expression levels were measured in 3 tissue sample pools of 10
freshly
slaughtered, healthy, lactating dairy cows: brain (cortex, thalamus,
hypothalamus), abomasum (fundus, corpus, antrum pylori), and intestine
pool (ileum, caecum, PLAC, colon). The 5-HT receptor expression was
quantified by normalization to the housekeeping gene
glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HT receptor
expression levels ranged from 0.0001% to 1% 5-HT/GAPDH. There was a
high variation of 5-HT receptor subtype mRNA expression within tissue
between receptor subtypes and within receptor subtypes between tissues.
In conclusion, accurate real-time PCR assays for quantitative analysis
of bovine 5-HT receptor subtype gene expression in disease models were
developed and validated.